DNA transfection

Silencing of PP2A: Human PP2Ac small interfering RNA (siRNA) was used to inhibit PP2Ac, which consists of a pool of three target-specific 19 to 25 nt siRNA. PP2A-Cα siRNA (h) is a pool of 3 different siRNA duplexes(5′ → 3′): A: Sense: GCAAAUCACCAGAUACAAAtt, Antisense: UUUGUAUCUGGUGAUUUGCtt; B: Sense: GAACUUGACGAUACUCUAAtt, Antisense: UUAGAGUAUCGUCAAGUUCtt; C: Sense: GGAUAGCAGCAAACAAUCAtt, Antisense: UGAUUGUUUGCUGCUAUCCtt. The nonsilencining control as a negative control consists of a scrambled sequence. For transfection, 2 × 10⁵ cells were seeded in culture plates and transfected with PP2Ac siRNA or scrambled siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) for 48 h before treatment with JS-K for 24 h.

PP2A overexpression: Cells were trypsinized, collected by centrifugation, and resuspended in regular medium. A single-cell suspension was then seeded at 2 × 10⁵ cells/well (12-well plates). Cells were infected or transfected with PP2A in serum-free medium the next day. For transfection, 2.5 μg of DNA was used for each transfection using lipofectamine 3000 reagent for 4 h. Then the infection medium was removed and replaced with complete medium for 48 h. Next, cells were treated with JS-K for 24 h to observe the expressions of proteins by Western blot analysis. Negative control plasmid was treated according the above methods.